The nematode, Caenorhabditis elegans has become an invaluable model organism for studying molecular and cellular functions. We are employing C. elegans to study human gene function as it relates to addiction. We are interested in how allelic variations in genes involved in the biosynthesis, transport, release, reuptake, signaling and catabolism of the neurotransmitter dopamine play a role in the susceptibilities to drugs of abuse such as methamphetamine. In humans and other mammals, dopamine is the primary neurotransmitter of the "reward pathway" which reinforces behaviors that are beneficial to the survival of an organism. It is the reward pathway that is most affected by drugs of abuse as they reinforce their consumption and associated behaviors. C. elegans has eight dopamine neurons that control specific movement and reproductive behaviors. This past year, we have adopted a swimming induced paralysis assay (SWIP) assay and developed a movement tracking assay to examine behavioral response to methamphetamine. We have also used this first year to acquire equipment and strains of C. elegans for our studies. We have hired a research associate with 7 years of experience working with C. elegans and associated molecular biology and transgenic techniques. During our summer students fellowship, she was able to acquire data on dose-dependent methamphetamine-induced changes in behavior. We are in the process of completing the study for publication. The presence of conserved genes involved in dopamine biology coupled with defined behaviors associated with these 8 dopamine neurons allows for testing human gene function in response to drugs of abuse. The ease of genetic manipulation in C. elegans allows us to replace the C. elegans gene such as the dopamine transporter with variants of human dopamine transporter. The "humanized" worms can then be analyzed for changes in drug response. Ultimately, by making genetically tagged versions of the human genes, we can screen for altered behavior after drug exposure and identify the combination of human alleles. We hypothesize that specific combinations of human gene variants or alleles can mediate altered responses to drugs of abuse and will indicate "vulnerabilities" to drugs such as methamphetamine or cocaine. From these studies, we hope use genetic based vulnerabilities to develop more individualized approaches to treating addiction. Towards these goals, we have acquired several variants of the human dopamine transporter with known differences in affinity and transport. We are in the process of generating transgenic worms on a worm dopamine transporter knockout background. In addition to studying gene function and drugs of abuse in the dopaminergic system, we can use models of dopaminergic neurodegeneration to study potential molecules important for dopaminergic neuron development, survival and regeneration of fibers. For example, we have been working on a conserved gene, mesencephalic astrocyte-derived neurotrophic factor (MANF) which was recently identified as a dopaminergic neurotrophic factor in mammals. From our studies in C elegans, we have been able to identify possible mechanisms of neuroprotection for this protein. This past year, we have 1) generated several transgenic animals that demonstrate the cellular location of MANF, 2) characterized MANF knockout worms, 3) generated and characterized antibodies for studying MANF in worms, and 4) identified a novel response to drugs that induce cellular stress. Some of our work was presented at the NIDA MiniSymposium at the 2009 Society for Neurosciene Meeting in Chicago. We are currently preparing the manuscript summarizing our findings of MANF. From our work with C.elegans and MANF, we will return to our mammalian models of Parkinsons disease and methamphetamine toxicity to examine new therapeutic approaches to treating the degeneration of dopaminergic neurons. In summary, we are able to employ C. elegans as a model for human gene function in dopaminergic neurons which are affected in addiction and Parkinsons disease.